Energy-Linked Binding of Pi Is Required for

R-subunit “noncatalytic” site-deficient TF1 R3β3γ complex and Fo‚F1 reconstituted from this complex are incapable of. ATP hydrolysis because o...
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14552

Biochemistry 2006, 45, 14552-14558

Energy-Linked Binding of Pi Is Required for Continuous Steady-State Proton-Translocating ATP Hydrolysis Catalyzed by Fo‚F1 ATP Synthase† Tatyana V. Zharova and Andrei D. Vinogradov* Department of Biochemistry, School of Biology, Moscow State UniVersity, Moscow 119992, Russian Federation ReceiVed July 28, 2006; ReVised Manuscript ReceiVed September 28, 2006

ABSTRACT: The presence of medium Pi (half-maximal concentration of 20 µM at pH 8.0) was found to be required for the prevention of the rapid decline in the rate of proton-motive force (pmf)-induced ATP hydrolysis by Fo‚F1 ATP synthase in coupled vesicles derived from Paracoccus denitrificans. The initial rate of the reaction was independent of Pi. The apparent affinity of Pi for its “ATPase-protecting” site was strongly decreased with partial uncoupling of the vesicles. Pi did not reactivate ATPase when added after complete time-dependent deactivation during the enzyme turnover. Arsenate and sulfate, which was shown to compete with Pi when Fo‚F1 catalyzed oxidative phosphorylation, substituted for Pi as the protectors of ATPase against the turnover-dependent deactivation. Under conditions where the enzyme turnover was not permitted (no ATP was present), Pi was not required for the pmf-induced activation of ATPase, whereas the presence of medium Pi (or sulfate) delayed the spontaneous deactivation of the enzyme which was induced by the membrane de-energization. The data are interpreted to suggest that coupled and uncoupled ATP hydrolysis catalyzed by Fo‚F1 ATP synthases proceeds via different intermediates. Pi dissociates after ADP if the coupling membrane is energized (no E‚ADP intermediate exists). Pi dissociates before ADP during uncoupled ATP hydrolysis, leaving the E‚ADP intermediate which is transformed into the inactive ADP(Mg2+)-inhibited form of the enzyme (latent ATPase).

Fo‚F1 synthases (ATPases) catalyze the production of ATP from ADP and Pi at the expense of the transmembrane proton (Na+)-motive force (pmf),1 generated by electron transfer reactions in the respiratory chains of mitochondria, chloroplasts, and bacterial plasma membranes. They are also capable of pmf generation with the use of energy from ATP hydrolysis; thus, the Fo‚F1 complex phenomenologically operates as the reversible energy-transducing device. The integral structures of Fo‚F1’s in all species studied so far are remarkably similar (1-4). The structures (5, 6), as related to rotary binding change mechanism (7-10) and kinetic properties (11) of F1-type ATPases, have been extensively discussed. When assayed either in the presence or in the absence of the ATP-regenerating system, F1 and Fo‚F1 ATPases exhibit complex time-dependent kinetics that are due to the relatively slow (as compared with the enzyme turnover) formation of the so-called ADP(Mg2+)-deactivated form, the phenomenon characteristic for all F1-type ATPases that have been studied. The fractional population of the ADP(Mg2+)-inhibited form in a certain preparation of F1 ATPase depends on a number † Τhis work was supported by the Russian Foundation for the Fundamental Studies (Grant 05-04-48809 to A.D.V.). * To whom correspondence should be addressed. Telephone and fax: 7-(495)-939-1376. E-mail: [email protected]. 1 Abbreviations: CCCP, carbonyl cyanide 3-chlorophenylhydrozone; CFo‚F1, EFo‚F1, MFo‚F1 PFo‚F1, and TFo‚F1, ATP synthase complexes in chloroplasts, Escherichia coli, bovine heart mitochondria, Paracoccus denitrificans, and thermophilic Bacillus PS3, respectively; NBD-Cl, 7-chloro-4-nitrobenzofurazan; pmf (∆µ˜ H+), difference between the electrochemical potential of the hydrogen ion at different sides of the coupling membrane.

of factors, such as the ATP/ADP ratio, pmf, free Mg2+ and inorganic phosphate concentrations, and the particular source of the enzyme (reviewed in ref 11). The most intriguing property of the ADP(Mg2+)-deactivated ATPase is that being inactive in ATP hydrolysis, it is fully competent in the ATP synthase activity (12, 13). Evidence that Fo‚F1 operates in the specific “hydrolase” or “synthase” modes depending on the presence of ATP, ADP, and pmf has been gradually accumulating (12-16). To create a model describing the reaction pathways in either the hydrolytic or synthetic direction of catalysis, quantitative information about interactions of the substrates (products), ATP, ADP, Pi, and Mg2+, with the enzyme is required. Numerous important studies of the nucleotide binding properties of F1 from different sources have been published and reviewed (see ref 17 and references cited therein). Less attention has been paid to Pi as the product (substrate) of ATP hydrolysis (synthesis), although its binding properties seem equally important for the understanding of the Fo‚F1 operation mechanism. Recently, we have shown that the presence of Pi in the assay mixture is required for the steadystate ATP hydrolysis catalyzed by tightly coupled vesicles derived from Paracoccus denitrificans plasma membrane (18). This paper describes further analysis of this phenomenon. To our knowledge, multiple effects of Pi on F1-type ATPases which may or may not be directly related to the mechanism of ATP synthesis have never been reviewed, and a brief account of the available data on this topic as outlined below seems to be appropriate for the introduction. Soluble bovine heart and Escherichia coli F1’s bind more than 1 mol of Pi per mole of enzyme with an apparent affinity

10.1021/bi061520v CCC: $33.50 © 2006 American Chemical Society Published on Web 11/04/2006

Binding of Pi to Fo‚F1 ATP Synthase of P. denitrificans in the micromolar range (19-21). Sulfate, sulfite, and chromate enhance Pi binding, whereas efrapeptin and azide, the specific inhibitors of ATP hydrolysis, inhibit Pi binding (19, 20). The removal of tightly bound nucleotides from MF1 prevents Pi binding, which is restored by preincubation of the enzyme with purine nucleotides (22). Pi bound to F1 is displaced by medium phosphate (19). Both the binding of Pi to and the dissociation of Pi from MF1 are slow processes that proceed on a minute time scale (19). The slow displacement of bound Pi is, however, greatly accelerated by ATP or ADP (23). Under the unisite ATP hydrolysis ([MF1] g [ATP]), the first-order rate constant for Pi and ADP release has been estimated to be 4 × 10-3 s-1 (24, 25). Pi (Ka ∼ 1 mM) was shown to stimulate the energy-promoted hydrolysis of ATP bound at a single high-affinity (Ka ∼ 1012 M-1) catalytic site of Fo‚F1 in coupled submitochondrial particles (26). Variable Km’s for Fo‚F1-catalyzed synthesis of ATP have been reported in the literature. These are 0.5-6 mM for MFo‚F1 (27-31), 0.6-3.5 mM for EFo‚F1 (32-34), 10 mM for TFo‚F1 (35), 0.25 mM for CFo‚F1 (36), and 0.15-0.35 mM for PFo‚F1 (37-39). The apparent affinity of Fo‚F1 for Pi in oxidative phosphorylation was shown to dependent on the rate of respiration and/or the steady-state magnitude of pmf (27, 29, 30, 33, 37, 39). An order of the substrates binding to F1 during ATP synthesis, if the same for different species, remains uncertain. Evidence for both ordered (31, 40) and random (38, 39) kinetic mechanisms has been published. Kayalar et al. (41) and Perez and Ferguson (38) have suggested a randomly ordered bi-uni reaction catalyzed by MFo‚F1 and PFo‚F1, respectively, whereas the compulsory ordered mechanisms where Pi binds first [MFo‚F1 (31)] or ADP binds first [CFo‚F1 (40)] have been suggested for oxidative and photophosphorylation, respectively. The fact that the exchange of medium Pi with H218O catalyzed by coupled submitochondrial particles in the presence of ATP and ADP is as sensitive to uncoupling as [32P]ATP exchange and net oxidative phosphorylation, whereas during uncoupled ATP hydrolysis, each Pi that is released contains more than one water 18O (intermediate exchange), was interpreted as evidence of the requirement of energy for binding of Pi to the catalytic site where the reversible energy-independent ATP formation takes place (42). The energy-independent enzyme-bound ATP formation from medium Pi has been directly demonstrated for CF1 (43), CFo‚F1 (44), MF1 (45), and TF1 (46). The half-maximal concentration of Pi required for this reaction was in the range of 20-40 mM. Pi was shown to increase the ATPase activity of rat liver mitochondria (47), submitochondrial particles derived therefrom (48, 49), or bovine heart soluble F1 (49) at 1-20 mM. This phenomenon is apparently due to the antagonistic effect of Pi on ADP(Mg2+)-induced deactivation of F1 or Fo‚F1 (50, 51). Indeed, Pi decreases the extent of high-affinity ADPspecific inhibitory binding in submitochondrial particles by 2 orders of magnitude (7 × 10-8 and 5 × 10-6 M in the absence and presence of 10 mM Pi, respectively) (50). Pi (Kd ) 1 mM) also increases the rate of activation of the ADP-inhibited MFo‚F1 in the absence of free Mg2+ (51). The R-subunit “noncatalytic” site-deficient TF1 R3β3γ complex and Fo‚F1 reconstituted from this complex are incapable of ATP hydrolysis because of instant entrapment of ADP in

Biochemistry, Vol. 45, No. 48, 2006 14553 their tight inhibitory site (14, 52). These mutant enzymes, however, catalyze continuous ATP hydrolysis in the presence of high (maximal rate at 150 mM) Pi concentrations (53). The trypsin cleavage of the -subunit of EF1 in the presence of ADP and Mg2+ was protected by Pi at the half-maximal concentration of 50 µM (54). Pi was also shown to protect ATPase in submitochondrial particles against irreversible inactivation by NBD-Cl at half-maximal efficiency reached at 0.2 mM (55). Recently, the “coupling” effect of Pi (along with ADP) at relatively low concentrations (half-maximal at 30 µM) on the proton translocating activity of Fo‚F1 ATPase in Rhodobacter capsulatus chromatophores has been described (56). Coupled plasma membrane vesicles derived from P. denitrificans do not catalyze ATP hydrolysis unless they are exposed to respiration-induced pmf (18, 57). As noted above, the presence of medium Pi was found to be indispensable for the pmf-induced proton-translocating ATP hydrolysis catalyzed by PFo‚F1 (18). Here we have examined the dependence of coupled ATPase on Pi. The data show that energy-dependent high-affinity Pi-binding site(s) must be occupied by Pi or by other anions (arsenate and sulfate) during the steady-state proton-translocating ATP hydrolysis to prevent the ADP(Mg2+)-induced inhibition of the enzyme. The minimal kinetic scheme describing coupled and uncoupled ATP hydrolysis catalyzed by Fo‚F1 ATP synthases is proposed and discussed with regard to the mechanism and reversibility of ATP hydrolysis. MATERIALS AND METHODS P. denitrificans (strain 1222) plasma membrane vesicles were prepared from a culture grown in the presence of succinate and nitrate (58). Under optimal conditions (57), the vesicles catalyzed the pmf-activated proton-translocating ATPase reaction [150 nmol min-1 (mg of protein)-1] and succinate-supported ATP synthesis [300 nmol min-1 (mg of protein)-1] at 30 °C. ATP hydrolysis and ATP synthesis were more than 90% sensitive to venturicidin and uncoupler, respectively. The rates of ATP hydrolysis or ATP synthesis were measured as small pH changes followed by Phenol Red responses (59). The level of inorganic phosphate was determined as described in ref 60. The standard reaction mixture for the ATPase assay was comprised of 0.25 M sucrose, 0.1 M KCl, 2.5 mM Hepes, 0.1 mM EDTA, 5.5 mM MgCl2, 2.5 mM succinate, 20 mM malonate, 2 mM ATP (potassium salts, pH 8.0), and 25 µM Phenol Red. ATP synthesis was assessed in the standard reaction mixture (except malonate was absent) supplemented with inorganic phosphate and ADP. The transmembrane potential was followed as Oxonol VI (1.5 µM) response at 624-602 nm. Oxygen consumption was assessed amperometrically with a platinum-coated oxygen-sensitive electrode. Other experimental details were described in our previous paper (18) or are given in the figure legends. Protein content was determined by the biuret procedure with bovine serum albumin as a standard. All fine chemicals were from Sigma, while other reagents were of the highest purity available from local suppliers. RESULTS The following background is relevant to the results on the steady-state pmf-induced ATPase activity of PFo‚F1. It has

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FIGURE 1: Effect of medium Pi on the time course of ATP hydrolysis. The vesicles (65 µg/mL) were added to the standard reaction mixture containing no malonate. After incubation for 2 min (coupled pmf-generating respiration), ATP hydrolysis was initiated by simultaneous addition of 1 mM ATP and 20 mM malonate (to eliminate succinate oxidation). (A) ATP hydrolysis (top panel, Phenol Red response) and membrane energization (bottom panel, Oxonol VI response) were followed in the standard reaction mixture containing Pi at the micromolar concentrations given on the curves. (B) Initial rate tracing of ATPase at higher sensitivity and time resolution. To ensure that the initial burst of Phenol Red response is, indeed, due to ATP hydrolysis, the control tracings where the vesicles were preincubated for 30 min with venturicidin, the specific inhibitor of prokaryotic ATPase (2 µg/mg of protein), are shown (top curves).

been shown that venturicidin-sensitive ATP hydrolytic activity of the vesicles as prepared is negligible unless pmf across the membrane is created by coupled oxidation of the respiratory substrate (succinate) (18). The proton-translocating ATP hydrolysis can then be seen if succinate oxidation is prevented by malonate. The ATPase activity thus revealed is stimulated by mild uncoupling (slight increase in the vectorial H+ cycling rate) and is inhibited if the membrane permeability for protons is sufficiently high to collapse pmf. The continuous steady-state ATPase activity is thus selfsupporting: pmf generated by Fo‚F1 ATPase is used to maintain the enzyme in its catalytically competent state. One should realize that any influence which decreases the rate of enzyme turnover is expected to cause two effects: a decrease in the overall enzyme activity and, as a result, a decrease in the fraction of active enzyme due to pmf reduction. Figure 1A shows the actual tracing of ATP hydrolysis and the membrane energization when the enzyme assays were conducted in the presence of different concentrations of Pi. ATP hydrolysis coupled with pmf generation was not seen in the absence of Pi in the assay, whereas zero-order ATPase and significant pmf generation, evident from the steady-state Oxonol response, proceeded in the presence of 2 mM Pi. Only transitory maintenance of pmf and a time-dependent decrease in the ATPase activity which was synchronous with a decrease in the magnitude of the Oxonol VI response were observed in the absence of Pi. The results shown in Figure 1A prompted us to follow the rate of the ATPase reaction at

Zharova and Vinogradov

FIGURE 2: Pi is unable to reactivate ATPase that is deactivated during ATP hydrolysis in the absence of Pi. ATP hydrolysis (top panel, Phenol Red response) and membrane energization (bottom panel, Oxonol VI response) were followed in the standard reaction mixture containing vesicles (73 µg/mL). After ATPase activity has declined (in the absence of Pi), 2 mM Pi was added where indicated followed by the addition of 35 µM NADH. Oxidation of NADH by the respiratory chain was accompanied by alkalinization of the medium according to the stoichiometric equation NADH + H+ + 1/ O f NAD+ + H O, which was evident from the Phenol Red 2 2 2 response (top panel) and transitory membrane energization (bottom panel). In the control experiments (not shown), NADH oxidation followed by the change in absorption at 340 nm was found to be synchronous with alkalinization of the medium followed by Phenol Red response. Neither ATPase activity nor the membrane energization was affected by the addition of NADH to the assay medium originally containing 2 mM Pi. The specific ATPase activities (nanomoles per minute per milligram of protein) are indicated by the numbers on the curves (top panel).

higher sensitive and time resolution. As seen in the traces shown in Figure 1B, the absence of ATPase activity in the samples containing no Pi was only apparent: the initial rates of ATP hydrolysis were independent of Pi, and a rapid decline of ATPase was seen in the absence of Pi. Determining whether the rapid deactivation of ATPase seen in the absence of Pi could be reversed was worth investigating. Figure 2 shows that the addition of 2 mM Pi (the concentration which is sufficient to support continuous zero-order proton-translocating ATP hydrolysis as depicted in Figure 1A) after the activity had declined did not induce ATPase activity. However, the activity was fully restored if the membranes were energized (as seen from the Oxonol VI response) by oxidation of a limited amount of NADH. After NADH has been consumed, rapid zero-order ATP hydrolysis was observed. Next we investigated the pmf dependence of the protecting effect of Pi against the enzyme deactivation. The timedependent ATPase activity measured at a fixed time (20 s) after initiation of the reaction by simultaneous addition of ATP and malonate was plotted as a function of Pi concentration in the assay mixture containing various concentrations of protonophoric uncoupler (CCCP) (Figure 3).

Binding of Pi to Fo‚F1 ATP Synthase of P. denitrificans

FIGURE 3: Effect of mild uncoupling on the protection of ATPase against deactivation by Pi. ATP hydrolysis was followed in the standard reaction mixture [containing vesicles (65 µg/mL)] and supplemented with Pi (concentrations are indicated on the abscissa). The specific ATPase activities (V) were estimated from the slopes of the reaction course curves 20 s after the reaction was initiated by the addition of ATP, malonate, and CCCP (simultaneously) as in Figure 1: (O) 0, (b) 0.3, (3) 1.0, (1) 2.0, and ([) 3.0 µM uncoupler. The specific initial rates (V) were stimulated upon uncoupling, and apparent rates estimated 20 s after time zero were normalized (V/V), where V corresponds to the rates seen at given uncoupler concentrations in the presence of 2 mM Pi. Please note that true initial rates were Pi-independent as shown in Figure 1.

Although this approach is not quantitative, the results that were obtained clearly show that the apparent affinity of Pi for its ATPase “protecting” site is pmf-dependent. The halfmaximal concentration of Pi (Pi,0.5) required for full ATPase activity was 15 µM and drastically increased upon partial uncoupling. It was noted that Pi,0.5 was variable depending on the coupling efficiency for different batches of the vesicle preparations. In attempts to clarify the variability of Pi,0.5 values, various ATPase assay media were tested. Surprisingly, no Pi requirement for the continuous zero-order ATP hydrolysis was found at all when KCl was omitted from the standard mixture. Further inspection revealed that the assay mixture (with or without KCl) contained 10 µM Pi as contamination from the vesicles (17 nmol/mg) and from potassium hydroxide used for the pH adjustment. In a KClfree assay mixture, this concomitant Pi (10 µM) was sufficient to protect ATPase against deactivation. From the data shown in Figure 3 and described above, it became evident that the energy-dependent high-affinity Pi binding is required to protect PFo‚F1 ATPase against the deactivation. This conclusion was corroborated by the partial uncoupling effect of potassium chloride: the respiratory control ratio determined with NADH as the substrate was 4.2 and 3.3 in the absence and presence of 0.1 M KCl, respectively. These results agree with those of Kell et al. (61), who have reported that the presence of KCl decreased the pmf of P. denitrificans vesicles respiring with NADH from 150 to 115 mV. Next, the specificity of Pi in its ATPase protective effect was studied. Arsenate (almost as efficient as Pi) and sulfate (less efficiently) were able to protect the enzyme activity (Figure 4), whereas acetate (10 mM) was ineffective. Sulfate was shown to compete with Pi when the rates of respiration-supported ATP synthesis were measured at saturating ADP concentrations. The apparent Km for Pi and the Ki for sulfate were 150 and 180 µM, respectively, if estimated assuming a simple competitive type of inhibition (results not shown). Although competition between Pi and sulfate in oxidative phosphorylation was evident, the doublereciprocal plots obtained in the presence of sulfate deviated

Biochemistry, Vol. 45, No. 48, 2006 14555

FIGURE 4: Arsenate and sulfate substitute for Pi in the protecting effect against the turnover-dependent deactivation of ATPase. The experimental conditions were the same as those described in the legend of Figure 3, except the uncoupler was not added.

FIGURE 5: Protective effect of Pi and sulfate on spontaneous deactivation of ATPase after de-energization of the membranes. Vesicles (80 µg/mL) were preincubated in the standard assay mixture containing no malonate for 2 min (energization of the membranes). Malonate (20 mM) was then added, and ATP hydrolysis was initiated by the addition of 2 mM ATP at the time indicated on the abscissa. The initial rates were measured as described in the legend of Figure 1A. Pi (2 mM) or sulfate (2 mM) was added to the assay medium where indicated. The circles depict data from the case in which no protecting anion was added. Pi and sulfate did not affect pmf-induced activation of ATPase. One hundred percent corresponds to a specific activity of 150 nmol min-1 (mg of protein)-1.

from a straight line in the high concentration range. This phenomenon was not further studied. The most likely reason for the deactivation of ATPase in the absence of added Pi during turnover as shown in Figure 1 seemed to originate from the widely studied although not completely understood so-called ADP(Mg2+)-induced inhibition, the phenomenon characteristic of all F1-type ATPases studied so far (62). In particular, latent PFo‚F1 ATPase was shown to be kinetically equivalent to the azide-trapped, ADP(Mg2+)-inhibited MFo‚F1 (18). Although Pi was unable to reactivate ATPase in the absence of pmf (Figure 2), we reasoned that activating anions (Pi and sulfate) should, at least partially, prevent the “spontaneous” (in fact, ADP reassociation-induced) transformation of active ATPase into its latent form. The results presented in Figure 5 show that this was, indeed, the case. Both Pi and SO4 delayed deactivation caused by elimination of pmf. DISCUSSION Numerous stable enzyme-substrate (products) complexes are expected to exist when Fo‚F1 catalyzes the steady-state ATP hydrolysis or (and) synthesis. At least five different forms of an ATPase having a single catalytic site [empty and ATP-, (ADP+Pi)-, ADP-, and Pi-loaded] are anticipated

14556 Biochemistry, Vol. 45, No. 48, 2006 at any given concentration of the substrate and products. This number becomes as large as 35 for F1-type ATPases (tree catalytic sites), assuming that bindings of ATP and Pi at a single catalytic site are mutually exclusive. What particular complexes are kinetically significant during the steady-state ATP hydrolysis and/or pmf-supported ATP synthesis remains to be established. We showed here, for the first time, the significant effect of Pi at low physiologically relevant concentrations (comparable with its apparent Km in oxidative phosphorylation) on the steady-state coupled ATPase activity. Pi drastically increases the steady-state ATPase activity catalyzed by pmfinduced ATPase with no effects on the “initial” rates (Figure 1). The time-dependent decrease in the rate of the ATPase reaction catalyzed by MFo‚F1 was shown to be a consequence of the turnover-dependent formation of the ADP(Mg2+)inhibited enzyme (62). The findings that Pi protects PFo‚F1 ATPase against spontaneous deactivation (Figure 5) and that this deactivation is, in fact, caused by the rebinding of ADP which dissociates during pmf-induced activation (18) suggest that the ADP(Mg2+)-inhibited form of the enzyme is the key intermediate responsible for the effect of Pi on the steadystate ATP hydrolysis. The kinetic scheme that consistently explains the findings reported in this paper is depicted in Figure 6. We propose that the major pathway of coupled protontranslocating ATP hydrolysis (when pmf is present) proceeds via steps 1-4 and Pi irreversibly leaves the enzyme after ADP dissociation. Essentially irreversible step 4 is likely to be the “stroke” step which is coupled with proton translocation. A predominance of steps 3 and 4 over steps 5 and 6 during coupled steady-state catalysis is presumably due to the inability of Pi to dissociate from the E‚(D+Pi) complex (energy-dependent tight binding of Pi to the E‚D complex). Pi rapidly and irreversibly dissociates (step 5) when the pmf decreases, thus leaving the E·D complex which then isomerizes into the dead-end E*‚D complex [step 7, formation of the ADP(Mg2+)-inhibited enzyme]. The reaction pathway via steps 1, 2, 5, and 6 operates when uncoupled Fo‚F1 or the soluble F1 catalyzes ATP hydrolysis. When pmf-induced, pmf-generating ATP hydrolysis proceeds in the presence of arsenate or sulfate (Figure 4), the ternary E‚(D+As) or E‚(D+SO4) complexes are formed, thus protecting the enzyme from isomerization into the dead-end ADP(Mg2+)inhibited species. We believe that the kinetic scheme shown in Figure 6 is qualitatively valid for all Fo‚F1-type ATPases. The differences in the kinetics of MFo‚F1, prokaryotic Fo‚F1, and CFo‚F1 ATPase activity seem likely to be quantitative rather than qualitative; the relative affinities of ADP and Pi for the catalytic sites of F1 ATPases and the rate constants for individual steps are evidently variable among the species. Pi does not inhibit coupled (this paper) or uncoupled (50) ATP hydrolysis catalyzed by F1-type ATPases. This phenomenon is hard, if not impossible, to explain if any reversibly operating ATPase/synthase model to be considered taking into account the fact that Pi is the substrate of pmfdriven ATP synthesis with an apparent Km in the millimolar range (see the introductory section). It seems unlikely that the reversal of steps 6, 5 (energydependent tight Pi binding), 2, and 1 is the reaction pathway during ATP synthesis. The evidence for random kinetics of

Zharova and Vinogradov

FIGURE 6: Kinetic scheme describing two reaction pathways for coupled and uncoupled ATP hydrolysis catalyzed by pmf-activated PFo‚F1 ATPase. T and D stand for ATP and ADP, respectively. The scheme does not attempt to assign individual kinetically distinct steps to the structural rearrangements of the enzyme during the catalysis such as the conformational change within the catalytic sites (open, closed, and half-closed conformations) and the particular position of the γ-subunit relative to the R-subunit-β-subunit interface nucleotide-binding sites. For the sake of simplicity, the enzyme is shown as a single catalytic unit and occupation of three (63) or two (64) catalytically competent sites by ATP, ADP, and Pi at each step is not specified. For example, one β-subunit may participate in step 1, whereas another β-subunit may catalyze the chemical transformation depicted as step 2 (binding change mechanism) (7). The participation of Mg2+ as nucleotide-Mg2+ complexes and free cations is also not specified. Steps 1-6 are the catalytic turnover steps. Step 5 likely corresponds to the release of Pi during unisite catalysis by mutated TF1 accompanied by the conformational change in the β-subunit (65). The dead-end ADP(Mg2+)-inhibited form (latent PFo‚F1 ATPase) is marked with an asterisk. This form is apparently intrinsically very stable in PFo‚F1 and additionally stabilized in MFo‚F1 if azide is present (step 8). An azide anion (pKa ) 4.7) may occupy a Pi-binding site, thus producing an E*‚(ADP+N3-) complex (66) structurally analogous to the enzyme-tightly bound ATP complex or ADP‚aluminum‚ fluoride (5) or ADP‚Vi‚fluoride (67) complexes. Slow (compared to the catalytic turnover) isomerization of the E‚D intermediate into the dead-end ADP(Mg2+)-inhibited form is shown as step 7. Step 5 is shown as pmf-dependent Pi binding and/or dissociation; i.e., Pi binds tightly or dissociates irreversibly when the coupling membrane is energized or de-energized, respectively. “Reversible” and “irreversible” steps are indicated by double-headed and singleheaded arrows, respectively. The term irreversible is used solely to indicate that no binding of a particular ligand occurs at physiologically relevant concentrations. The scheme is an extension of the originally proposed kinetic mechanism of uncoupled ATP hydrolysis catalyzed by F1-type ATPase (62, 68) as adapted for the more physiologically relevant proton translocation-coupled reaction.

ATP synthesis catalyzed by PFo‚F1, the same species that used in these studies, has been presented (39). The deadend ADP(Mg2+)-inhibited form, which is stabilized by azide in MFo‚F1 (68) or intrinsically stable in PFo‚F1 (18), is an unlikely intermediate of ATP synthesis because the latter process is azide-insensitive (12, 14) and, more significantly, the mutated TF1 incapable of ATP-promoted activation of the inhibited form is fully active in the ATP synthase reaction (14). The apparent affinity of Pi for the energized PFo‚F1 is much higher [K0.5 ) 8-15 µM (this paper)] than the apparent Km for Pi in the ATP synthesis reaction (this paper and ref 38). Taken together, these facts agree with the proposal that different states (conformations) of Fo‚F1 catalyze pmfgenerating ATPase or pmf-driven ATP synthase reactions (12-16). Further detailed kinetic studies of Fo‚F1 operating in the ATP synthase mode, particularly with respect to the order of the substrates binding to the enzyme, are required to determine whether the intermediates of uncoupled or coupled ATP hydrolysis catalyzed by F1 are different from those which exist during coupled ATP synthesis. The molecular mechanism for the ATPase-ATP synthase switch remains unclear. A likely candidate is the -subunit for which

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